The medial entorhinal cortex (MEC) contributes to navigation and episodic memory, essential cognitive functions degraded in many degenerative and psychiatric disorders. A key to MEC function was provided by the discovery of grid cells, which fire on the vertices of a set of hexagonal lattices tessellating space. The grid cell system has been hypothesized to perform path integration during navigation and to be a map of the spatial environment. Because of the striking regularity of their firing fields, grid cells have generated widespread theoretical interest, and numerous models have been proposed to explain how grids are formed, how they are organized in microcircuits, and how they might use idiothetic (self motion) information to path integrate. The grid cell system therefore offers the opportunity to study a cognitively meaningful neural computation at a mechanistic level. Here we leverage recent technical advances, including virtual reality methods for rodents previously developed in our lab, to examine the intracellular, microcircuit, and integrative properties of gri cells in three aims: 1.) Current grid cell models can reproduce hexagonal lattice firing patterns but they predict different intracellular membrane potential time courses that reflect different underlying cellular or network mechanisms. To test these predictions, in Aim 1, we will take advantage of head-fixed navigation enabled by our virtual reality system to make intracellular recordings from grid cells during behavior. Statistical analysis will be performed on the membrane voltage time series to examine if characteristic features such as ramps and theta oscillations are present and if they correlate with the location of the firing fields. For example,we will examine if theta oscillation amplitude is larger in firing fields, and if theta frequency increases with mouse velocity, as predicted by theta interference models of grid cells. 2.) Grid cells are not identical, but have different scales and phase shifts that may reflect distinct functional modules. Consistent with this idea, converging evidence points to the existence of anatomically defined clusters of cells in MEC. To delineate the link between functional modules and anatomical clusters, in Aim 2 we will use cellular-resolution two-photon calcium imaging during virtual navigation to provide the first measurements of spatial organization, at the microcircuit scale, of identified grid cells in MEC. In particular, we will map the relationship between grid cell properties (spatial scale and phase) and cytochrome oxidase rich patches, and determine whether there are sharp breaks in spatial scale along the dorsoventral axis. 3.) Grid cells are thought to perform path integration, an idea that dominates the current thinking about the functional role of the MEC. In Aim 3 we will use virtual reality to control all sensory cues providing information about position in order to rigorously test the path integration hypothesis. Together, these aims should advance our understanding of the single-cell, microcircuit, and computational properties of grid cells. PUBLIC HEALTH RELEVANCE: Using electrophysiological and behavioral methods, we will examine the mechanisms by which the medial entorhinal cortex establishes a metric representation of the environment. Many diseases are associated with functional disruption of the entorhinal cortex and hippocampus, such as Alzheimer's disease, schizophrenia, and temporal lobe epilepsy. The proposed experiments will address the fundamental mechanisms of neural circuit dynamics, generating insight into the normal operation of the entorhinal cortex and how it may be altered in disease.